Dan forms condensates in neuroblasts and regulates nuclear architecture and progenitor competence in vivo

Genome organization is thought to underlie cell type specific gene expression, yet how it is regulated in progenitors to produce cellular diversity is unknown. In Drosophila, a developmentally-timed genome reorganization in neural progenitors terminates competence to produce early-born neurons. These events require downregulation of Distal antenna (Dan), part of the conserved pipsqueak DNA-binding superfamily. Here we find that Dan forms liquid-like condensates with high protein mobility, and whose size and subnuclear distribution are balanced with its DNA-binding. Further, we identify a LARKS domain, a structural motif associated with condensate-forming proteins. Deleting just 13 amino acids from LARKS abrogates Dan’s ability to retain the early-born neural fate gene, hunchback, in the neuroblast nuclear interior and maintain competence in vivo. Conversely, domain-swapping with LARKS from known phase-separating proteins rescues Dan’s effects on competence. Together, we provide in vivo evidence for condensate formation and the regulation of progenitor nuclear architecture underlying neuronal diversification.


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Antibodies
Antibodies used This study contains no human research participants.
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All experiments were performed following field standards described in numerous publications whose results have been validated by multiple groups.These are all referenced in the manuscript.At least three independent animals that were carefully matched by genotype and by developmental stage were included for both the control and experiment.Competence experiments include analyzing 10-15 neuroblast lineages per embryo and at least three embryos per genotype and stage.Controls were performed in parallel and confirmed to match results of several previous publications, indicating high reproducibility and consistency in results and methodology.
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For embryo competence experiments, Dan misexpression using UAS-mediated constructs showed similar results between Dan without epitope tags and Dan with epitope tags (myc, GFP), as well as UAS-Dan inserted into different chromosomal locations, indicating reproducibility of observations both within this study and compared to previously published results.For DNA FISH experiments, YW (wild type) embryo analyses were included, to validate reproducibility and consistency with previously published results.
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